The useful lifetime of proteins, particularly those developed for pharmaceutical administration, is extended by storage as frozen, lyophilized or refrigerated aqueous compositions. However, upon thawing, frozen non-lyophilized monomeric proteins have a tendency to form polymeric aggregates. Although the formation of a multivalent compound may be desirable in specific situations, unwanted aggregation is disadvantageous in a monomeric protein composition.
Antibodies represent a specific class of proteins generated by the immune system to provide a molecule capable of complexing with an invading molecule, termed an antigen. Natural antibodies have two identical antigen-binding sites, both of which are specific to a particular antigen. The antibody molecule "recognizes" the antigen by complexing its antigen-binding sites with areas termed epitopes. The epitopes fit into the conformational architecture of the antigen-binding sites of the antibody, enabling the antibody to bind to the antigen.
The antibody molecule is composed of two identical heavy and two identical light chains, held together by disulfide bonds. Covalent interchain and intrachain bonding serves to stabilize the various chains of antibody molecules.
The light chain comprises one variable region (termed V.sub.L) and one constant region (C.sub.L), while the heavy chain comprises one variable region (termed V.sub.H) and three constant regions (C.sub.H 1, C.sub.H 2 and C.sub.H 2). Pairs of regions associate to form discrete structures. The light-chain variable region, V.sub.L, and the heavy-chain variable region, V.sub.H, of a particular antibody molecule have specific amino acid sequences that allow the antigen-binding site to assume a conformation that binds to the antigen epitope recognized by that particular antibody. More specifically, the light and heavy chain variable regions, V.sub.L and V.sub.H associate to form an "F.sub.v " area which contains the antigen-binding site.
Cleavage of the naturally-occurring antibody molecule with a proteolytic enzyme generates fragments which retain their antigen binding site. Fragments of this type, commonly known as Fab (for Fragment, antigen binding) are composed of the C.sub.L, V.sub.L, C.sub.H 1 and V.sub.H regions of the antibody, wherein the light chain and the fragment of the heavy chain are covalently linked by a disulfide linkage.
Recent advances in immunobiology, recombinant DNA technology, and computer science have allowed the creation of single polypeptide chain molecules that bind antigen. These single-chain antigen-binding molecules contain only the variable domains of the antibody, incorporating a linker polypeptide to bridge the variable regions, V.sub.L and V.sub.H, into a single, monomeric polypeptide chain, the "sF.sub.v." A computer-assisted method for linker design is described more particularly in U.S. Pat. No. 4,704,692. A description of the theory and production of single-chain antigen-binding proteins is found in U.S. Pat. Nos. 4,946,778 and 5,260,203. The single-chain antigen-binding proteins produced under the process recited in U.S. Pat. Nos. 4,946,778 and 5,260,203 have binding specificity and affinity substantially similar to that of the corresponding Fab fragment. Several different single-chain molecules that can successfully bind antigen have now been constructed using a variety of peptide linkers. For a review, see, Whitlow and Filpula, Methods: Compan. Methods Enzymol. 2:97-105 (1991).
Many of the early single-chain F.sub.v products were insoluble aggregates, requiring solubilization under strong denaturing solutions, followed by renaturing and proper refolding before they could manifest single-chain antigen binding ability. See, e.g., Buchner et al., Bio/Technol. 9:157 (1991) and Buchner et al., Analyt. Biochem. 205:263 (1992). Thus, it has been found to be particularly advantageous to provide for the expression of a single-chain antigen-binding molecule as a soluble product, which can be purified directly from the periplasmic fraction without the need for in vitro manipulation and refolding. See, Sawyer et al., Protein Engineering 7:1401 (1994).
However, if the soluble, monomeric protein composition forms aggregates during frozen storage of the non-lyophilized product, or as a result of the freeze/thaw process, a heterogeneous, unstable composition may result in which immunoreactivity may be diminished. At the very least, disadvantageous aggregation of the protein monomers as a result of storage instabilities may require additional purification steps (including denaturation and refolding) to restore the protein to a composition of homogeneous monomers. Biological consistency and stability are essential for most clinical applications of a monomeric, single-chain antigen-binding protein composition.
Studies have been made, at both the academic and clinical levels, of the decomposition mechanisms of proteins. As a result, the art has recognized that different proteins exhibit highly variable inactivation responses. For example, R. L. Levine has reported in J. Biol. Chem. 258:11828 (1983) an analysis of the effects of 24 amino acids and sulfhydryl compounds as stabilizers to inhibit glutamine synthetase degradation in a system containing oxygen, ascorbate and trace metal. Levine disclosed that only cysteine and histidine showed significant activity in preventing loss of activity for the enzyme, whereas some of the compounds tested actually stimulated the inactivation reaction. For instance, the inactivation of the enzyme creatine kinase by ascorbate has been shown to be stimulated by histidine.
Several authors have described methods for stabilizing a lyophilized protein composition. For example, in U.S. Pat. No. 4,496,537, Kwan describes the enhanced storage stability of lyophilized alpha-type interferon formulations, by incorporating glycine or alanine prior to lyophilization. The resulting lyophilized formulations can be stored without loss of activity for more than six months at 20.degree. C. before reconstitution with water.
An abstract of Japanese Patent Application 59-181224 discloses an enhanced stability for interferons having added an amino acid and, optionally, human serum albumin before freeze drying. Yasushi et al. disclose in U.S. Pat. No. 4,645,830, that interleukin-2 is stabilized against loss of activity during freezing, lyophilization or storage, by formulating the composition to include human serum albumin, a reducing compound or both, and by adjusting the pH to between 3 and 6. According to Yasushi et al., the interleukin-2 formulation may also contain an amino acid, particularly glycine, a monosaccharide, and/or a sugar alcohol.
To avoid the inconvenience of reconstituting a lyophilized product and because of the potential for introducing an error during such procedures, other authors have analyzed methods for stabilizing a refrigerated, aqueous protein composition. An abstract of Japanese Patent Application 57-26587 describes the stabilization of ascorbic acid oxidase by adding one or more of the following: arginine, lysine, histidine and borates. In addition, in U.S. Pat. No. 4,806,524, Kawaguchi et al. describe the stabilization of either freeze-dried or aqueous erythropoietin formulations against decomposition, by adding one or more of the following: polyethylene glycol, proteins, sugars, amino acids, inorganic salts, organic salts and sulfur-containing reducing agents. Furthermore, in U.S. Pat. No. 4,777,043, Bennett et al. report that an increased solubility and stability is obtained when human tissue plasminogen activator is formulated to contain arginine, as the protonated cation "argininium ion."
More recently, Patel, in U.S. Pat. No. 5,358,708, has disclosed a method for increasing the storage stability of an aqueous formulation containing a protein component selected from among the group consisting of interferons, granulocyte-macrophage colony-stimulating factors and interleukins, by the addition of a stabilizing amount of methionine, histidine or mixtures thereof. Patel reports that without such treatment, aqueous protein formulations typically have short useful storage lives after reconstitution, even when stored at low temperatures (e.g., 5.degree. C.).
Finally, certain authors have described particular methods for the stabilization of protein compositions designed specifically for pharmaceutical applications. According to Jensen in U.S. Pat. No. 3,950,513, the solubility and stability of plasmin solutions for parenteral administration are enhanced by the addition of physiologically non-toxic amino acids. While, in Technical Report No. 10, entitled "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," J. Parenteral Sci. & Technol. 42:S12-S13, Supplement 1988, Y-C. J. Wang and M. A. Hanson review the use of amino acids to stabilize parenteral formulations of proteins and peptides.
Recent publications have reported that besides the protein structure itself, the surrounding solvent shell is of crucial importance for the stability and dynamic properties of the protein composition. See Muller et al. Biochemistry 33:6221 (1994). Moreover, even the role of water in protein aggregation and protein reactions has received considerable attention (Rand, Science 256:618 (1992); Colombo et al., Science 256:655 (1992).
A number of investigators have reported the spontaneous aggregation of sF.sub.v s. Weidner et al., J. Biol. Chem. 267:10281 (1992) observed aggregates of the 4-4-20/212 sF.sub.v ; Mezes et al., Third Annual IBC Intern'l Conf. on Antibody Engineering, San Diego, Calif. (1992) reported aggregates of the CC49/205c sF.sub.v ; Adams et al., Cancer Res. 53:4026 (1993) and Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444 (1993) described divalent sF.sub.v of anti-c-erbB-2 sF.sub.v ; and Whitlow et al, Protein Eng. 7:1017 (1994) investigated heterodimers of the CC49 and 4-4-20 sF.sub.v s.
However, until the discovery of the present invention, there remained a long-felt need in the art for a method of stabilizing frozen, non-lyophilized, monomeric protein compositions to inhibit or prevent unwanted aggregation of the soluble polypeptide molecules, particularly when the composition is exposed to repeated freeze/thaw cycles. Therefore, the invention of the presently formulated, stabilized protein composition, its method of preparation, and pharmaceutically-acceptable compositions prepared thereby will significantly advance the art by ensuring a stable supply of such proteins as consistent, essentially homogeneous monomers.